The standard for the prevention of rejection in transplant patients consists in immunosuppressive regimens including a calcineurin inhibitor such as cyclosporine (CsA) or tacrolimus (TAC). However, the optimal balance between a too strong or a too weak immunosuppression is difficult to achieve following transplantation. An inadequate immunosuppression can provoke a transplant rejection and, on the other hand, an excessive immunosuppression facilitates the development of severe complications such as infections or lymphoproliferative disorders. The usual practice consists, for the physicians, in establishing the treatment with fixed doses, then in adjusting the drug doses according to their blood levels or, even more frequently, according to the occurrence of either an acute rejection or adverse drug event. Unfortunately, this strategy is associated with a frequent failure as illustrated by the fact that 30 to 50% of the patients develop acute rejection after transplantation.
A pharmacodynamic approach, based on the measurement of the effect of the drugs on their cellular targets, has been developed with the aim to reduce the incidence and severity of acute rejection. This pharmacodynamic approach is based on the measurement of the activity of calcineurin (CN), a calcium-calmodulin-dependent phosphatase. CN is part of the family of the serine/threonine phosphatase enzyme including PP1, PP2A, PP2B or calcineurin and PP2C (Ingebritsen & Cohen, 1983).
Current calcineurin assays are generally performed in cell extracts obtained from blood mononuclear cells (PBMC, peripheral blood mononuclear cells) from transplant patients (Fruman et al, 1996; Sanquer et al, 2004; Fukudo et al, 2005). These tests are based on the dephosphorylation of a phosphorylated substrate of calcineurin in the presence of calcium and calmodulin which are essential to the activity of the enzyme (Pallen & Wang, 1983). The addition of okadaic acid at a concentration of 500 nM (that inhibits all the phosphatases except CN) avoids the potential cross-reactivity for the phosphorylated substrate.
An especially preferred substrate is the phosphorylated peptide RII (P-RII), derived from the regulatory subunit of protein kinase A (PKA, Donella-Deana et al, 1994). This peptide is characterized by the following sequence: DLDVPIPGRFDRRVpSVAAE (SEQ ID No:1).
In the different assays described so far, the P-RII peptide can be labelled or not, and the labelling is either performed with a radioelement (32P) or a fluorophore.
Most methods used for measuring CN phosphatase activity are based on the detection of free phosphate released during the enzymatic reaction. These methods use radiometric measurements in the case of P-RII peptide labelled with 32P (Fruman et al, 1996; Koefoed-Nielsen et al, 2004). They use spectrophotometric measurements in the case of unlabelled R-RII peptide. The latter requires the implementation of a second reaction with green malachite to color the free inorganic phosphate (Sellar et al, 2006).
More specific methods based on the direct measurement of the formation of the dephosphorylated peptide RII (DP-RII) by CN have been developed. These methods were originally based on liquid chromatography coupled with UV detection (Enz et al, 1994; Sanquer et al, 2004). These methods were developed for unlabelled P-RII peptide. More recently, a fluorimetric method was described to measure CN activity by using P-RII peptide labelled with a fluorophore (Roberts et al, 2008).